Project Summary/Abstract: Cochlear tonotopy describes how different frequencies are represented along the apex to base axis of the organ of Corti. A small number of about 3500 sensory cells constitute each of the four rows of hair cells in the human organ of Corti, which in essence resolves the wide range of frequencies necessary for speech discrimination and communication. For a long time, its limited number of cells and the difficult access hampered investigations on the mechanism determining the fine-tuned molecular architecture of this sensory organ. The overall aim of this study is to overcome limitations caused by the paucity of the target cell population and to establish novel strategies to investigate the highly dynamic process of organ of Corti development using single cell RNA based technology in combination with spatial and temporal transcriptomic analyses. Research proposed in this project will resolve embryonic gene expression gradients along the tonotopic axis using spatial trajectory reconstruction methods from whole transcriptome RNA sequencing data obtained from single murine organ of Corti cells. Gradients depending on sonic hedgehog signaling may potentially confer spatial information consecutively translated in the tonotopic organization of the sensory organ. The aim of this proposal is to test this hypothesis by comparing knock out and over-expression models of sonic hedgehog signaling with respect to changes in previously identified gene expression gradients in the embryo. Embryonic manipulations of sonic hedgehog signaling potentially disturb cochlear tonotopy permanently. Analyzing transcriptional profiles and physiological properties of cochlear hair cells after the onset of hearing will test this hypothesis by comparing loss of function, gain of function, and control specimen.